Axenfeld-Rieger Syndrome Sequencing Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1995 COL4A1 81408 Add to Order
CYP1B1 81404
FOXC1 81479
PAX6 81479
PITX2 81479
SH3PXD2B 81479
Full Panel Price* $1790.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1995 Genes x (6) $1790.00 81404, 81408, 81479(x4) Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information. If you would like to order a subset of these genes contact us to discuss pricing.

For Sanger Sequencing click here.
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Approximately 25%-60% of Axenfeld-Rieger syndrome cases are due to FOXC1 or PITX2 pathogenic variants (Tümer and Bach-Holm 2009; Reis et al. 2012; Alward 2000). In one study, two out of 30 Axenfeld-Rieger syndrome patients (6.7%) carried SH3PXD2B sequence variants (Mao et al. 2012). Due to the phenotypic and genotypic heterogeneity, clinical sensitivity of the other genes in this panel is difficult to predict.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 CYP1B1$690.00 81479 Add to Order
FOXC1$690.00 81479
PAX6$690.00 81479
PITX2$690.00 81479
Full Panel Price* $840.00
Pricing Comment

# of Genes Ordered

Total Price













Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Axenfeld-Rieger syndrome (ARS) is a rare, highly penetrant disorder characterized by varying degrees of eye anterior segment anomalies with systemic malformations such as dental hypoplasia and a protuberant umbilicus (Hjalt and Semina 2005; Berry et al. 2006; Waldron et al. 2010; Tümer and Bach-Holm 2009; Chang et al. 2012). Dental abnormalities in this syndrome help in diagnosis and to distinguish ARS from other eye anterior segment abnormalities. Early diagnosis of ARS from its dento-facial and systemic features is essential in treating or preventing the most serious consequence of ARS (O’Dwyer and Jones 2005). The major clinical concern is high risk of developing open-angle glaucoma, which represents the main challenge in terms of treatment. Approximately 50% of ARS affected patients develop glaucoma (Shields et al. 1985; Alward 2000; Hjalt and Semina 2005; Chang et al. 2012). ARS affected patients also need surveillance and management of sensorineural hearing loss, and cardiac, endocrinological, craniofacial and orthopaedic defects (Chang et al. 2012).

Rieger syndrome (RIEG) and Peters anomaly both are in the anterior chamber cleavage group of anomalies and are considered to be variations of a single developmental disorder (the ARS group) (Reese and Ellsworth 1966). RIEG is characterized by malformations of the eyes, teeth, and umbilicus; whereas Peters anomaly displays only ocular features (Amendt et al. 2000; Doward et al 1999).


Pathogenic variants in the FOXC1 or PITX2 genes cause autosomal dominant Axenfeld-Rieger syndrome (ARS). Linkage studies identified four chromosomal loci (4q25, 6p25, 13q14 and 16q24) that are associated with ARS and related or overlapping phenotypes. The genes that have been identified on chromosomes 4q25 and 6p25 are PITX2 (the pituitary homeobox 2 gene) and FOXC1 (the forkhead box C1 gene, also known as FKHL7) respectively (Alward 2000; Lines et al. 2002; Tümer and Bach-Holm 2009). PITX2 and FOXC1 are two transcription factor genes which are expressed throughout eye ontogeny (Lines et al. 2002). The genes at 13q14 and 16q24 have not yet been discovered.

PITX2 encodes a Paired-like homeodomain transcription factor (PITX2), which plays a critical role in cell proliferation, differentiation, hematopoiesis and organogenesis (Huang et al. 2009). Also, PITX2 integrates retinoic acid and canonical Wnt signaling during eye anterior segment development (Gage et al. 2008; Gage and Zacharias 2009).

The product of the FOXC1 gene (FOXC1) is reported to maintain homeostasis in trabecular meshwork (TM) cells by regulating genes that play an important role in stress response (Ito et al. 2014; Paylakhi et al. 2013). TM helps in regulating intraocular pressure by acting as a drainage structure for aqueous humor (Tamm 2009). Pathogenic mutations in FOXC1 significantly decrease the TM cell viability and subsequently contribute to the development of glaucoma.

A Genotype-Phenotype correlation study indicated that patients with PITX2 pathogenic variants have a more severe prognosis for glaucoma as compared to FOXC1 pathogenic variants. Within FOXC1 pathogenic variants, patients with FOXC1 duplications are at higher risk for the development of glaucoma, which emphasizes the importance of genetic testing. It’s been reported that PITX2 and FOXC1 interact with each other, which is essential for the regulation of common downstream target genes within specific cell lineages (Berry et al. 2006). Also, co-inheritance of PITX2 and FOXC1 pathogenic variants has been reported in a family which segregated with the disease and showed variable phenotypic expression. The most severely affected individual had pathogenic variants in both genes, whereas single heterozygous variants caused milder ARS phenotypes (Kelberman et al. 2011). So far, about 80 and 100 ARS causative variants have been identified in the PITX2 and FOXC1 genes, respectively (Human Gene Mutation Database).

Pathogenic variants in COL4A1 also cause autosomal dominant ARS (Sibon et al. 2007). Pathogenic variants in SH3PXD2B cause autosomal recessive Frank-ter Haar syndrome. SH3PXD2B analysis in Axenfeld-Rieger syndrome patients identified several rare variants. However, the pathogenicity of these has not been well documented (Mao et al. 2012).

Pathogenic variants in PAX6 and CYP1B1 have shown to be associated with  Peters anomaly (Hanson et al. 1994; Vincent et al. 2001).

A wide variety of causative variants (missense, nonsense, splicing, small deletions and insertions) have been reported in Axenfeld-Rieger syndrome and overlapping phenotypes -associated genes including large deletions/duplications and complex genomic rearrangements in few genes (FOXC1, PAX6, CYP1B1 and PITX2) (Human Gene Mutation Database).

See individual gene test descriptions for more information on molecular biology of gene products.

Testing Strategy

For this Next Generation (NextGen) panel, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

Indications for Test

All patients with symptoms suggestive of Axenfeld–Rieger syndrome, Rieger syndrome and Peters anomaly are candidates.


Official Gene Symbol OMIM ID
COL4A1 120130
CYP1B1 601771
FOXC1 601090
PAX6 607108
PITX2 601542
SH3PXD2B 613293
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

PITX2- Related Disorders via PITX2 Gene
Aniridia via The PAX6 Gene
FOXC1-Related Disorders via the FOXC1 Gene
Glaucoma Sequencing Panel
Primary Congenital Glaucoma via the CYP1B1 Gene


Genetic Counselors
  • Alward WL. 2000. American Journal of Ophthalmology. 130: 107-15. PubMed ID: 11004268
  • Amendt BA. et al. 2000. Cellular and Molecular Life Sciences : Cmls. 57: 1652-66. PubMed ID: 11092457
  • Berry FB. et al. 2006. Human Molecular Genetics. 15: 905-19. PubMed ID: 16449236
  • Chang TC. et al. 2012. The British Journal of Ophthalmology. 96: 318-22. PubMed ID: 22199394
  • Doward W. et al. 1999. Journal of Medical Genetics. 36: 152-5. PubMed ID: 10051017
  • Gage PJ. et al. 2008. Developmental Biology. 317: 310-24. PubMed ID: 18367164
  • Gage PJ., Zacharias AL. 2009. Developmental Dynamics : an Official Publication of the American Association of Anatomists. 238: 2149-62. PubMed ID: 19623614
  • Hanson IM. et al. 1994. Nature Genetics. 6: 168-73. PubMed ID: 8162071
  • Hjalt TA., Semina EV. 2005. Expert Reviews in Molecular Medicine. 7: 1-17. PubMed ID: 16274491
  • Huang Y. et al. 2009. Febs Letters. 583: 638-42. PubMed ID: 19174163
  • Human Gene Mutation Database (Bio-base).
  • Ito YA. et al. 2014. Cell Death & Disease. 5: e1069. PubMed ID: 24556684
  • Kelberman D. et al. 2011. Human Mutatation. 32: 1144–52. PubMed ID: 21837767
  • Lines MA. et al. 2002. Human Molecular Genetics. 11: 1177–84. PubMed ID: 12015277
  • Mao M. et al. 2012. Molecular Vision. 18: 705-13.  PubMed ID: 22509100
  • O'Dwyer E.M., Jones D.C. 2005. International Journal of Paediatric Dentistry. 15: 459-63. PubMed ID: 16238657
  • Paylakhi SH. et al. 2013. Experimental Eye Research. 111: 112-21. PubMed ID: 23541832
  • Reese AB., Ellsworth RM. 1966. Archives of Ophthalmology. 75: 307-18. PubMed ID: 5948260
  • Reis L.M. et al. 2012. European Journal of Human Genetics. 20: 1224-33. PubMed ID: 22569110
  • Shields MB. et al. 1985. Survey of Ophthalmology. 29: 387-409. PubMed ID: 3892740
  • Sibon I. et al. 2007. Annals of Neurolog. 62: 177–84. PubMed ID: 17696175
  • Tamm ER. 2009. Experimental Eye Research. 88: 648-55. PubMed ID: 19239914
  • Tümer Z, Bach-Holm D. 2009. European Journal of Human Genetics : Ejhg. 17: 1527-39. PubMed ID: 19513095
  • Vincent A. et al. 2001. Journal of Medical Genetics. 38: 324-6.  PubMed ID: 11403040
  • Waldron JM. et al. 2010. Special Care in Dentistry : Official Publication of the American Association of Hospital Dentists, the Academy of Dentistry For the Handicapped, and the American Society For Geriatric Dentistry. 30: 218-22. PubMed ID: 20831741
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NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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